Reagents and methods for specific binding assays

A composition comprising a complex of avidin and a molecule having two biotinyl radicals connected to one another by a chain at least 16 atoms in length, said chain being conjugated to a member of a specific binding pair ("sbp member") wherein said chain is not part of said sbp member, said chain having a 1,3-disubstituted aromatic ring as part of said chain and said biotinyl radicals are substantially exclusively bivalently bound to said single molecule of avidin.

In an assay for an analyte wherein there is employed a reagent system comprising an avidin reagent and a biotin reagent, the improvement comprising using as the biotin reagent a composition comprising two biotinyl radicals connected by a chain at least 16 atoms in length having a 1,3-disubstituted aromatic ring as part of said chain, wherein said biotinyl radicals substantially exclusively bivalently bind to a single molecule of avidin, wherein a member of a specific binding pair ("sbp member") is conjugated to said chain, and wherein none of the atoms of said chain is part of said sbp member.

A kit comprising in packaged combination:

• (a) an avidin reagent for use in a specific binding assay and
• (b) a compound consisting of a bis-biotin comprised of two biotinyl radicals connected by a chain at least 16 atoms in length, said bis-biotin being conjugated to a member of a specific binding pair ("sbp member") wherein said chain is not part of said sbp member and said chain has a 1,3-disubstituted aromatic ring as part of said chain such that said biotinyl radicals substantially exclusively bivalently bind to a single molecule of avidin.

A method of preparing a bis-biotinylated conjugate of a member of a specific binding pair ("sbp member") for use in a specific binding assay comprising the step of reacting said sbp member with a molecule containing two biotinyl radicals connected by a chain of at least 16 atoms having a 1,3-disubstituted aromatic ring as part of said chain, said chain comprising an attaching group for attaching said molecule to said sbp member, and wherein said biotinyl radicals substantially exclusively bivalently bind with a single molecule of avidin.

A complex of (i) a compound that is bis-biotin comprising two biotinyl radicals connected together by a chain of at least 16 atoms including a 1,3-disubstituted aromatic ring as part of said chain, said bis-biotin being conjugated to a member of a specific binding pair ("sbp member") wherein said chain is not part of said sbp member and (ii) avidin, wherein said biotinyl radicals are substantially exclusively bivalently bound to a single molecule of avidin and wherein said complex remains substantially intact when stored for two weeks at 37.degree. in a 10.sup.-6 M biotin solution.

A composition comprising avidin complexed with a bis-biotin having a chain, connecting the two biotinyl radicals, of at least 16 atoms including a 1,3-disubstituted aromatic ring as part of said chain wherein said complexes have only one or two bis-biotin molecules substantially exclusively bivalently bound to each avidin molecule.

A compound comprising two biotinyl radicals covalently bound together by a linking group comprising a chain of atoms at least 16 atoms in length including a 1,3-disubstituted aromatic ring as part of said chain wherein said biotinyl radicals substantially exclusively bivalently bind with a single molecule of avidin, said chain having an attaching group bound thereto, wherein, when a solution containing (i) less than or equal to 10.sup.-8 M of a 1:1 mole:mole complex of said compound and fully active avidin and (ii) 10.sup.-6 M free biotin is incubated at 37.degree. C. for at least 14 days, said complex exhibits less than 1% dissociation.

A compound that is a bis-biotin comprising two biotinyl radicals connected by a chain at least 16 atoms in length including a 1,3-disubstituted aromatic ring as part of said chain wherein said biotinyl radicals substantially exclusively bivalently bind with a single molecule of avidin, said chain comprising a functionality reactive with a corresponding functionality of a protein, said functionality and said corresponding functionality further characterized in that their reaction with each other does not substantially affect the activity of said protein.